Phosphorus fluoride exchange (PFEx): a new frontier in bioconjugation, enzyme inhibition, proximity-induced target-binding and fluorescence probes

Phosphorus fluoride Exchange (PFEx) chemistry has recently emerged as a powerful addition to the click chemistry toolbox, introducing P(V)-centred electrophiles as versatile handles for covalent bond formation in chemical biology. Distinguished by an unusual balance between hydrolytic stability and latent reactivity, P(V)-F functionalities enable highly orthogonal selective, proximity-driven transformations with nucleophilic residues in complex biological environments. The article presents for the first time PFEx chemistry, particularly analyzing its expanding important role in chemical biology and medicinal chemistry. We trace conceptual development of PFEx chemistry from established click paradigms, including copper-catalysed azide-alkyne cycloadditions (CuAAC) and sulfur-fluoride exchange (SuFEx). Their unique reactivity, chemoselectivity and orthogonality are highlighted. Recent advances in the genetic encoding of phosphoramidofluoridate-containing unnatural amino acids, such as PFY and PFK, are discussed, enabling site-specific and catalyst-free covalent modification of proteins both in-vitro and in mammalian cells through proximity-enabled mechanisms. The ability of PFEx to engage a broad spectrum of nucleophilic residues and to generate stable covalent linkages underscores its versatility as a bioconjugation strategy. In addition, enzyme-guided PFEx processes are highlighted as an emerging approach for the development of potent irreversible inhibitors, exemplified by targeting enzymes such as serine hydrolases and glutamine synthetase. The utility of fluorophosphonate-based electrophiles in activity-based protein profiling is also examined, particularly for the selective labelling and functional identification of enzymes like SH and GS. Collectively, PFEx chemistry represents a rapidly advancing Frontier at the interface of synthetic organic chemistry, chemical biology and drug discovery, offering new opportunities to precisely modulate biomolecular function through covalent mechanisms.